Fibroblast growth factor 15

ABSTRACT

Disclosed is a human Fibroblast growth factor-15 polypeptide and DNA(RNA) encoding such polypeptide. Also provided is a procedure for producing such polypeptide by recombinant techniques. Also disclosed are methods for utilizing such polypeptide for stimulating re-vascularization, for treating wounds and prevent neuronal damage. Antagonists against such polypeptides and their use as a therapeutic to prevent abnormal cellular proliferation, hyper-vascular diseases and epithelial lens cell proliferation are also disclosed. Diagnostic methods for detecting mutations in the coding sequence and alterations in the concentration of the polypeptides in a sample derived from a host are also disclosed.

[0001] This invention relates to newly identified polynucleotides,polypeptides encoded by such polynucleotides, the use of suchpolynucleotides and polypeptides, as well as the production of suchpolynucleotides and polypeptides. More particularly, the polypeptide ofthe present invention have been putatively identified as fibroblastgrowth factor/heparin binding growth factor, hereinafter referred to as“FGF-15”. The invention also relates to inhibiting the action of suchpolypeptides.

[0002] Fibroblast growth factors are a family of proteins characteristicof binding to heparin and are, therefore, also called heparin bindinggrowth factors (HBGF). Expression of different members of these proteinsare found in various tissues and are under particular temporal andspatial control. These proteins are potent mitogens for a variety ofcells of mesodermal, ectodermal, and endodermal origin, includingfibroblasts, corneal and vascular endothelial cells, granulocytes,adrenal cortical cells, chondrocytes, myoblasts, vascular smooth musclecells, lens epithelial cells, melanocytes, keratinocytes,oligodendrocytes, astrocytes, osteoblasts, and hematopoietic cells.

[0003] Each member has functions overlapping with others and also hasits unique spectrum of functions. In addition to the ability tostimulate proliferation of vascular endothelial cells, both FGF-1 and 2are chemotactic for endothelial cells and FGF-2 has been shown to enableendothelial cells to penetrate the basement membrane. Consistent withthese properties, both FGF-1 and 2 have the capacity to stimulateangiogenesis. Another important feature of these growth factors is theirability to promote wound healing. Many other members of the FGF familyshare similar activities with FGF-1 and 2 such as promoting angiogenesisand wound healing. Several members of the FGF family have been shown toinduce mesoderm formation and to modulate differentiation of neuronalcells, adipocytes and skeletal muscle cells.

[0004] Other than these biological activities in normal tissues, FGFproteins have been implicated in promoting tumorigenesis in carcinomasand sarcomas by promoting tumor vascularization and as transformingproteins when their expression is deregulated.

[0005] The FGF family presently consists of eight structurally-relatedpolypeptides: basic FGF, acidic FGF, int 2, hst 1/k-FGF, FGF-5, FGF-6,keratinocyte growth factor, AIGF (FGF-8) and recently a glia-activatingfactor has been shown to be a novel heparin-binding growth factor whichwas purified from the culture supernatant of a human glioma cell line(Miyamoto, M. et al., Mol. and Cell. Biol., 13(7):4251-4259 (1993). Thegenes for each have been cloned and sequenced. Two of the members, FGF-1and FGF-2, have been characterized under many names, but most often asacidic and basic fibroblast growth factor, respectively. The normal geneproducts influence the general proliferation capacity of the majority ofmesoderm and neuroectoderm-derived cells. They are capable of inducingangiogenesis in vivo and may play important roles in early development(Burgess, W. H. and Maciag, T., Annu. Rev. Biochem., 58:575-606,(1989)).

[0006] Many of the above-identified members of the FGF family also bindto the same receptors and elicit a second message through binding tothese receptors.

[0007] A eukaryotic expression vector encoding a secreted form of FGF-1has been introduced by gene transfer into porcine arteries. This modeldefines gene function in the arterial wall in vivo. FGF-1 expressioninduced intimal thickening in porcine arteries 21 days after genetransfer (Nabel, E. G., et al., Nature, 362:844-6 (1993)). It hasfurther been demonstrated that basic fibroblast growth factor mayregulate glioma growth and progression independent of its role in tumorangiogenesis and that basic fibroblast growth factor release orsecretion may be required for these actions (Morrison, R. S., et al., J.Neurosci. Res., 34:502-9 (1993)).

[0008] Fibroblast growth factors, such as basic FGF, have further beenimplicated in the growth of Kaposi's sarcoma cells in vitro (Huang, Y.Q., et al., J. Clin. Invest., 91:1191-7 (1993)). Also, the cDNA sequenceencoding human basic fibroblast growth factor has been cloned downstreamof a transcription promoter recognized by the bacteriophage T7 RNApolymerase. Basic fibroblast growth factors so obtained have been shownto have biological activity indistinguishable from human placentalfibroblast growth factor in mitogenicity, synthesis of plasminogenactivator and angiogenesis assays (Squires, C. H., et al., J. Biol.Chem., 263:16297-302 (1988)).

[0009] U.S. Pat. No. 5,155,214 discloses substantially pure mammalianbasic fibroblast growth factors and their production. The amino acidsequences of bovine and human basic fibroblast growth factor aredisclosed, as well as the DNA sequence encoding the polypeptide of thebovine species.

[0010] Newly discovered FGF-9 has around 30% sequence similarity toother members of the FGF family. Two cysteine residues and otherconsensus sequences in family members were also well conserved in theFGF-9 sequence. FGF-9 was found to have no typical signal sequence inits N terminus like those in acidic and basic FGF. However, FGF-9 wasfound to be secreted from cells after synthesis despite its lack of atypical signal sequence FGF (Miyamoto, M. et al., Mol. and Cell. Biol.,13(7):4251-4259 (1993). Further, FGF-9 was found to stimulate the cellgrowth of oligodendrocyte type 2 astrocyte progenitor cells, BALB/c3T3,and PC-12 cells but not that of human umbilical vein endothelial cells(Naruo, K., et al., J. Biol. Chem., 266:2857-2864 (1993).

[0011] Basic FGF and acidic FGF are potent modulators of cellproliferation, cell motility, differentiation, and survival and act oncell types from ectoderm, mesoderm and endoderm. These two FGFs, alongwith KGF and AIGF, were identified by protein purification. However, theother four members were isolated as oncogenes., expression of which wasrestricted to embryogenesis and certian types of cancers. FGF-9 wasdemonstrated to be a mitogen against glial cells. Members of the FGFfamily are reported to have oncogenic potency. FGF-9 has showntransforming potency when transformed into BALB/c3T3 cells (Miyamoto,M., et al., Mol. Cell. Biol., 13(7):4251-4259 (1993).

[0012] Androgen induced growth factor (AIGF), also known as FGF-8, waspurified from a conditioned medium of mouse mammary carcinoma cells(SC-3) simulated with testosterone. AIGF is a distinctive FGF-likegrowth factor, having a putative signal peptide and sharing 30-40%homology with known members of the FGF family. Mammalian cellstransformed with AIGF shows a remarkable stimulatory effect on thegrowth of SC-3 cells in the absence of androgen. Therefore, AIGFmediates androgen-induced growth of SC-3 cells, and perhaps other cells,since it is secreted by the tumor cells themselves.

[0013] The polypeptide of the present invention has been putativelyidentified as a member of the FGF family as a result of amino acidsequence homology with other members of the FGF family.

[0014] In accordance with one aspect of the present invention, there areprovided novel mature polypeptides as well as biologically active anddiagnostically or therapeutically useful fragments, analogs andderivatives thereof. The polypeptides of the present invention are ofhuman origin.

[0015] In accordance with another aspect of the present invention, thereare provided isolated nucleic acid molecules encoding the polypeptidesof the present invention, including mRNAs, DNAs, cDNAs, genomic DNA, aswell as antisense analogs thereof and biologically active anddiagnostically or therapeutically useful fragments thereof.

[0016] In accordance with still another aspect of the present invention,there are provided processes for producing such polypeptides byrecombinant techniques through the use of recombinant vectors, such ascloning and expression plasmids useful as reagents in the recombinantproduction of the polypeptides of the present invention, as well asrecombinant prokaryotic and/or eukaryotic host cells comprising anucleic acid sequence encoding a polypeptide of the present invention.

[0017] In accordance with a further aspect of the present invention,there is provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, for screening for agonistsand antagonists thereto and for therapeutic purposes, for example,promoting wound healing for example as a result of burns and ulcers, toprevent neuronal damage due to neuronal disorders and promote neuronalgrowth, and to prevent skin aging and hair loss, to stimulateangiogenesis, mesodermal induction in early embryos and limbregeneration.

[0018] In accordance with yet a further aspect of the present invention,there are provided antibodies against such polypeptides.

[0019] In accordance with yet another aspect of the present invention,there are provided antagonists against such polypeptides and processesfor their use to inhibit the action of such polypeptides, for example,in the treatment of cellular transformation, for example, tumors, toreduce scarring and treat hyper-vascular diseases.

[0020] In accordance with another aspect of the present invention, thereare provided nucleic acid probes comprising nucleic acid molecules ofsufficient length to specifically hybridize to a polynucleotide encodinga polypeptide of the present invention

[0021] In accordance with yet another aspect of the present invention,there are provided diagnostic assays for detecting diseases orsusceptibility to diseases related to mutations in a nucleic acidsequence of the present invention and for detecting over-expression ofthe polypeptides encoded by such sequences.

[0022] In accordance with another aspect of the present invention, thereis provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, for in vitro purposesrelated to scientific research, synthesis of DNA and manufacture of DNAvectors.

[0023] These and other aspects of the present invention should beapparent to those skilled in the art from the teachings herein.

[0024] The following drawings are meant only as illustrations ofspecific embodiments of the present invention and are not meant aslimitations in any manner.

[0025]FIG. 1 depicts the cDNA sequence and corresponding deduced aminoacid sequence of FGF-15.

[0026]FIG. 2 illustrates the amino acid sequence homology, betweenFGF-15 and the other FGF family members. Conserved amino acids arereadily ascertainable.

[0027] In accordance with one aspect of the present invention, there areprovided isolated nucleic acids molecules (polynucleotides) which encodefor the mature polypeptide having the deduced amino acid sequence ofFIG. 1 (SEQ ID NOS:2) or for the mature polypeptide encoded by the cDNAof the clone deposited as ATCC Deposit No. 97146 on May 12, 1995.

[0028] The polynucleotide encoding FGF-15 of this invention wasdiscovered initially in a cDNA library derived from human adrenal tumortissue. It is structurally related to all members of the fibroblastgrowth factor family and contains an open reading frame encoding apolypeptide of 252 amino acids. Among the top matches are: 1) 41%identity and 66% sequence similarity to human FGF-9 over a stretch of129 amino acids; and 2) 37% identity and 59% similarity to human KGFover a region of 88 amino acids.

[0029] The FGF/HBGF family signature, GXLX(S,T,A,G)X6(D,E)CXFXE isconserved in the polypeptide of the present invention, (X means anyamino acid residue; (D,E) means either D or E residue; X6 means any 6amino acid residues).

[0030] The polynucleotide of the present invention may be in the form ofRNA or in the form of DNA, which DNA includes cDNA, genomic DNA, andsynthetic DNA. The DNA may be double-stranded or single-stranded. Thecoding sequence which encodes the mature polypeptide may be identical tothe coding sequence shown in FIG. 1 (SEQ ID NO:1) or that of thedeposited clone or may be a different coding sequence, as a result ofthe redundancy or degeneracy of the genetic code, encodes the same,mature polypeptide as the DNA of FIG. 1, (SEQ ID NO:1) or the depositedcDNA.

[0031] The polynucleotides which encodes for the mature polypeptide ofFIG. 1 (SEQ ID NO:2) or for the mature polypeptides encoded by thedeposited cDNA(s) may include: only the coding sequence for the maturepolypeptide; the coding sequence for the mature polypeptide andadditional coding sequence such as a leader or secretory sequence or aproprotein sequence; the coding sequence for the mature polypeptide (andoptionally additional coding sequence) and non-coding sequence, such asintrons or non-coding sequence 5′ and/or 3′ of the coding sequence forthe mature polypeptide.

[0032] Thus, the term “polynucleotide encoding a polypeptide”encompasses a polynucleotide which includes only coding sequence for thepolypeptide as well as a polynucleotide which includes additional codingand/or non-coding sequence.

[0033] The present invention further relates to variants of thehereinabove described polynucleotides which encode for fragments,analogs and derivatives of the polypeptides having the deduced aminoacid sequence of FIG. 1 (SEQ ID NO:2) or the polypeptides encoded by thecDNA(s) of the deposited clone(s). The variants of the polynucleotidemay be a naturally occurring allelic variant of the polynucleotide or anon-naturally occurring variant of the polynucleotide.

[0034] Thus, the present invention includes polynucleotides encoding thesame mature polypeptide as shown in FIG. 1 (SEQ ID NO:2) or the samemature polypeptides encoded by the cDNA(s) of the deposited clone(s) aswell as variants of such polynucleotides which variants encode for afragment, derivative or analog of the polypeptide of FIG. 1 (SEQ IDNO:2) or the polypeptides encoded by the cDNA(s) of the depositedclone(s). Such nucleotide variants include deletion variants,substitution variants and addition or insertion variants.

[0035] As hereinabove indicated, the polynucleotide may have a codingsequence which is a naturally occurring allelic variant of the codingsequence shown in FIG. 1 (SEQ ID NO:1) or of the coding sequence of thedeposited clone(s). As known in the art, an allelic variant is analternate form of a polynucleotide sequence which may have asubstitution, deletion or addition of one or more nucleotides, whichdoes not substantially alter the function of the encoded polypeptides.

[0036] The present invention also includes polynucleotides, wherein thecoding sequence for the mature polypeptides may be fused in the samereading frame to a polynucleotide sequence which aids in expression andsecretion of a polypeptide from a host cell, for example, a leadersequence which functions as a secretory sequence for controllingtransport of a polypeptide from the cell. The polypeptide having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the mature form of the polypeptide. Thepolynucleotides may also encode for a proprotein which is the matureprotein plus additional 5′ amino acid residues. A mature protein havinga prosequence is a proprotein and is an inactive form of the protein.Once the prosequence is cleaved an active mature protein remains.

[0037] Thus, for example, the polynucleotides of the present inventionmay encode for a mature protein, or for a protein having a prosequenceor for a protein having both a prosequence and a presequence (leadersequence).

[0038] The polynucleotides of the present invention may also have thecoding sequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

[0039] The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

[0040] Fragments of the full length FGF-15 gene may be used as ahybridization probe for a cDNA library to isolate the full length geneand to isolate other genes which have a high sequence similarity to thegene or similar biological activity. Probes of this type preferably haveat least 30 bases and may contain, for example, 50 or more bases. Theprobe may also be used to identify a cDNA clone corresponding to a fulllength transcript and a genomic clone or clones that contain thecomplete FGF-15 gene including regulatory and promotor regions, exons,and introns. An example of a screen comprises isolating the codingregion of the FGF-15 gene by using the known DNA sequence to synthesizean oligonucleotide probe. Labeled oligonucleotides having a sequencecomplementary to that of the gene of the present invention are used toscreen a library of human cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

[0041] The present invention further relates to polynucleotides whichhybridize to the hereinabove-described sequences if there is at least70%, preferably at least 90%, and more preferably at least 95% identitybetween the sequences. The present invention particularly relates topolynucleotides which hybridize under stringent conditions to thehereinabove-described polynucleotides. As herein used, the term“stringent conditions” means hybridization will occur only if there isat least 95% and preferably at least 97% identity between the sequences.The polynucleotides which hybridize to the hereinabove describedpolynucleotides in a preferred embodiment encode polypeptides whicheither retain substantially the same biological function or activity asthe mature polypeptide encoded by the cDNAs of FIG. 1 (SEQ ID NO:1) orthe deposited cDNA(s).

[0042] Alternatively, the polynucleotide may have at least 20 bases,preferably 30 bases, and more preferably at least 50 bases whichhybridize to a polynucleotide of the present invention and which has anidentity thereto, as hereinabove described, and which may or may notretain activity. For example, such polynucleotides may be employed asprobes for the polynucleotide of SEQ ID NO:1, for example, for recoveryof the polynucleotide or as a diagnostic probe or as a PCR primer.

[0043] Thus, the present invention is directed to polynucleotides havingat least a 70% identity, preferably at least 90% and more preferably atleast a 95% identity to a polynucleotide which encodes the polypeptideof SEQ ID NO:2 as well as fragments thereof, which fragments have atleast 30 bases and preferably at least 50 bases and to polypeptidesencoded by such polynucleotides.

[0044] The deposit(s) referred to herein will be maintained under theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the purposes of Patent Procedure. These deposits areprovided merely as a convenience and are not an admission that a depositis required under 35 U.S.C. § 112. The sequence of the polynucleotidescontained in the deposited materials, as well as the amino acid sequenceof the polypeptides encoded thereby, are incorporated herein byreference and are controlling in the event of any conflict with thedescription of sequences herein. A license may be required to make, useor sell the deposited materials, and no such license is hereby granted.

[0045] The present invention further relates to an FGF polypeptide whichhas the deduced amino acid sequence of FIG. 1 (SEQ ID NO:2) or which hasthe amino acid sequence encoded by the deposited cDNA(s), as well asfragments, analogs and derivatives of such polypeptides.

[0046] The terms “fragment,” “derivative” and “analog” when referring tothe polypeptide of FIG. 1 (SEQ ID NO:2) or those encoded by thedeposited cDNA(s), means polypeptides which retains essentially the samebiological function or activity as such polypeptides. Thus, an analogincludes a proprotein which can be activated by cleavage of theproprotein portion to produce an active mature polypeptide.

[0047] The polypeptides of the present invention may be recombinantpolypeptides, natural polypeptides or synthetic polypeptides, preferablyrecombinant polypeptides.

[0048] The fragment, derivative or analog of the polypeptide of FIG. 1(SEQ ID NO:2) or that encoded by the deposited cDNA(s) may be (i) one inwhich one or more of the amino acid residues are substituted with aconserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code, or (ii) one in which one or moreof the amino acid residues includes a substituent group, or (iii) one inwhich the mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of the maturepolypeptide or a proprotein sequence. Such fragments, derivatives andanalogs are deemed to be within the scope of those skilled in the artfrom the teachings herein.

[0049] The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

[0050] The term “isolated” means that the material is removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or DNA or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotide could be part of a vector and/or such polynucleotide orpolypeptide could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

[0051] The polypeptides of the present invention include the polypeptideof SEQ ID NO:2 (in particular the mature polypeptide) as well aspolypeptides which have at least 70% similarity (preferably at least a70% identity) to the polypeptide of SEQ ID NO:2 and more preferably atleast a 90% similarity (more preferably at least a 90% identity) to thepolypeptide of SEQ ID NO:2 and still more preferably at least a 95%similarity (still more preferably a 95% identity) to the polypeptide ofSEQ ID NO:2 and also include portions of such polypeptides with suchportion of the polypeptide generally containing at least 30 amino acidsand more preferably at least 50 amino acids.

[0052] As known in the art “similarity” between two polypeptides isdetermined by comparing the amino acid sequence and its conserved aminoacid substitutes of one polypeptide to the sequence of a secondpolypeptide.

[0053] Fragments or portions of the polypeptides of the presentinvention may be employed for producing the corresponding full-lengthpolypeptide by peptide synthesis; therefore, the fragments may beemployed as intermediates for producing the full-length polypeptides.Fragments or portions of the polynucleotides of the present inventionmay be used to synthesize full-length polynucleotides of the presentinvention.

[0054] The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

[0055] Host cells may be genetically engineered (transduced ortransformed or transfected) with the vectors of this invention which maybe, for example, a cloning vector or an expression vector. The vectormay be, for example, in the form of a plasmid, a viral particle, aphage, etc. The engineered host cells can be cultured in conventionalnutrient media modified as appropriate for activating promoters,selecting transformants or amplifying the FGF genes. The cultureconditions, such as temperature, pH and the like, are those previouslyused with the host cell selected for expression, and will be apparent tothe ordinarily skilled artisan.

[0056] The polynucleotide of the present invention may be employed forproducing a polypeptide by recombinant techniques. Thus, for example,the polynucleotide sequence may be included in any one of a variety ofexpression vehicles, in particular vectors or plasmids for expressing apolypeptide. Such vectors include chromosomal, nonchromosomal andsynthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids;phage DNA; yeast plasmids; vectors derived from combinations of plasmidsand phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus,and pseudorabies. However, any other vector or plasmid may be used aslong as they-are replicable and viable in the host.

[0057] The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease sites by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

[0058] The DNA sequence in the expression vector is operatively linkedto an appropriate expression control sequence(s) (promoter) to directmRNA synthesis. As representative examples of such promoters, there maybe mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

[0059] In addition, the expression vectors preferably contain a gene toprovide a phenotypic trait for selection of transformed host cells suchas dihydrofolate reductase or neomycin resistance for eukaryotic cellculture, or such as tetracycline or ampicillin resistance in E. coli.

[0060] The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence,may be employed to transform an appropriate host to permit the host toexpress the protein. As representative examples of appropriate hosts,there may be mentioned: bacterial cells, such as E. coli, Salmonellatyphimurium, Streptomyces; fungal cells, such as yeast; insect cells,such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COSor Bowes melanoma; adenoviruses; plant cells, etc. The selection of anappropriate host is deemed to be within the scope of those skilled inthe art from the teachings herein.

[0061] More particularly, the present invention also includesrecombinant constructs comprising one or more of the sequences asbroadly described above. The constructs comprise a vector, such as aplasmid or viral vector, into which a sequence of the invention has beeninserted, in a forward or reverse orientation. In a preferred aspect ofthis embodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pBS, phagescript, psiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a,pNH46a (Stratagene); pTRC99A, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, PXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

[0062] Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

[0063] In a further embodiment, the present invention relates to hostcells containing the above-described construct. The host cell can be ahigher eukaryotic cell, such as a mammalian cell, or a lower eukaryoticcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell. Introduction of the construct into the hostcell can be effected by calcium phosphate transfection, DEAE-Dextranmediated transfection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, 1986)).

[0064] The constructs in host cells can be used in a conventional mannerto produce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

[0065] Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, (Cold SpringHarbor, N.Y., 1989), the disclosure of which is hereby incorporated byreference.

[0066] Transcription of a DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp, that act on a promoter to increase itstranscription. Examples include the SV40 enhancer on the late side ofthe replication origin (bp 100 to 270), a cytomegalovirus early promoterenhancer, a polyoma enhancer on the late side of the replication origin,and adenovirus enhancers.

[0067] Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), α factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation, initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

[0068] Useful expression vectors for bacterial use are constructed byinserting a structural DNA sequence encoding a desired protein togetherwith suitable translation, initiation and termination signals inoperable reading phase with a functional promoter. The vector willcomprise one or more phenotypic selectable markers and an origin ofreplication to ensure maintenance of the vector and to, if desirable,provide amplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

[0069] As a representative but nonlimiting example, useful expressionvectors for bacterial use can comprise a selectable marker and bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of the well known cloning vector pBR322(ATCC 37017). Such commercial vectors include, for example, pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec,Madison, Wis., USA). These pBR322 “backbone” sections are combined withan appropriate promoter and the structural sequence to be expressed.

[0070] Following transformation of a suitable host strain and growth ofthe host strain to an appropriate cell density, the selected promoter isderepressed by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

[0071] Cells are typically harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification.

[0072] Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents.

[0073] Various mammalian cell culture systems can also be employed toexpress recombinant protein. Examples of mammalian expression systemsinclude the COS-7 lines of monkey kidney fibroblasts, described byGluzman, Cell, 23:175 (1981), and other cell lines capable of expressinga compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and accentorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 viralgenome, for example, SV40 origin, early promoter, enhancer, splice, andpolyadenylation sites may be used to provide the required nontranscribedgenetic elements.

[0074] The polypeptide of the present invention may be recovered andpurified from recombinant cell cultures by methods used heretofore,including ammonium sulfate or ethanol precipitation, acid extraction,anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxyapatite chromatography and lectin chromatography.Protein refolding steps can be used, as necessary, in completingconfiguration of the mature protein. Finally, high performance liquidchromatography (HPLC) can be employed for final purification steps.

[0075] The polypeptide of the present invention may be a naturallypurified product, or a product of chemical synthetic procedures, orproduced by recombinant techniques from a prokaryotic or eukaryotic host(for example, by bacterial, yeast, higher plant, insect and mammaliancells in culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated with mammalian or other eukaryotic carbohydrates or may benon-glycosylated. Polypeptides of the invention may also include aninitial methionine amino acid residue.

[0076] The polypeptide of the present invention, as a result of theability to stimulate vascular endothelial cell growth, may be employedin treatment for stimulating re-vascularization of ischemic tissues dueto various disease conditions such as thrombosis, arteriosclerosis, andother cardiovascular conditions. These polypeptide may also be employedto stimulate angiogenesis and limb regeneration.

[0077] The polypeptide may also be employed for treating wounds due toinjuries, burns, post-operating tissue repair, and ulcers since they aremitogenic to various cells of different origins, such as fibroblastcells and skeletal muscle cells, and therefore, facilitate the repair orreplacement of damaged or diseased tissue.

[0078] The polypeptide of the present invention may also be employedstimulate neuronal growth and to treat and prevent neuronal damage whichoccurs in certain neuronal disorders or neuro-degenerative conditionssuch as Alzheimer's disease, Parkinson's disease, and AIDS-relatedcomplex. FGF-15 has the ability to stimulate chondrocyte growth,therefore, they may be employed to enhance bone and periodontalregeneration and aid in tissue transplants or bone grafts.

[0079] The polypeptide of the present invention may be also be employedto prevent skin aging due to sunburn by stimulating keratinocyte growth.

[0080] The FGF-15 polypeptide may also be employed for preventing hairloss, since FGF family members activate hair-forming cells and promotesmelanocyte growth. Along the same lines, the polypeptides of the presentinvention may be employed to stimulate growth and differentiation ofhematopoietic cells and bone marrow cells when used in combination withother cytokines.

[0081] The FGF-15 polypeptide may also be employed to maintain organsbefore transplantation or for supporting cell culture of primarytissues.

[0082] The polypeptide of the present invention may also be employed forinducing tissue of mesodermal origin to differentiate in early embryos.

[0083] In accordance with yet a further aspect of the present invention,there is provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, for in vitro purposesrelated to scientific research, synthesis of DNA, manufacture of DNAvectors and for the purpose of providing diagnostics and therapeuticsfor the treatment of human disease.

[0084] This invention provides a method for identification of thereceptors for the polypeptides of the present invention. The genesencoding the receptor can be identified by numerous methods known tothose of skill in the art, for example, ligand panning and FACS sorting(Coligan, et al., Current Protocols in Immun., 1(2), Chapter 5, (1991)).Preferably, expression cloning is employed wherein polyadenylated RNA isprepared from a cell responsive to the polypeptides, for example, NIH3T3cells which are known to contain multiple receptors for the FGF familyproteins, and SC-3 cells, and a cDNA library created from this RNA isdivided into pools and used to transfect COS cells or other cells thatare not responsive to the polypeptides. Transfected cells which aregrown on glass slides are exposed to the the polypeptide of the presentinvention, after they have been labelled. The polypeptides can belabeled by a variety of means including iodination or inclusion of arecognition site for a site-specific protein kinase.

[0085] Following fixation and incubation, the slides are subjected toauto-radiographic analysis. Positive pools are identified and sub-poolsare prepared and re-transfected using an iterative sub-pooling andre-screening process, eventually yielding a single clones that encodesthe putative receptor.

[0086] As an alternative approach for receptor identification, thelabeled polypeptides can be photoaffinity linked with cell membrane orextract preparations that express the receptor molecule Cross-linkedmaterial is resolved by PAGE analysis and exposed to X-ray film. Thelabeled complex containing the receptors of the polypeptides can beexcised, resolved into peptide fragments, and subjected to proteinmicrosequencing. The amino acid sequence obtained from microsequencingwould be used to design a set of degenerate oligonucleotide probes toscreen a cDNA library to identify the genes encoding the putativereceptors.

[0087] This invention provides a method of screening compounds toidentify those which modulate the action of the polypeptide of thepresent invention. An example of such an assay comprises combining amammalian fibroblast cell, a the polypeptide of the present invention,the compound to be screened and ³[H] thymidine under cell cultureconditions where the fibroblast cell would normally proliferate. Acontrol assay may be performed in the absence of the compound to bescreened and compared to the amount of fibroblast proliferation in thepresence of the compound to determine if the compound stimulatesproliferation by determining the uptake of ³[H] thymidine in each case.The amount of fibroblast cell proliferation is measured by liquidscintillation chromatography which measures the incorporation of ³[H]thymidine. Both agonist and antagonist compounds may be identified bythis procedure.

[0088] In another method, a mammalian cell or membrane preparationexpressing a receptor for a polypeptide of the present invention isincubated with a labeled polypeptide of the present invention in thepresence of the compound. The ability of the compound to enhance orblock this interaction could then be measured. Alternatively, theresponse of a known second messenger system following interaction of acompound to be screened and the FGF-15 receptor is measured and theability of the compound to bind to the receptor and elicit a secondmessenger response is measured to determine if the compound is apotential agonist or antagonist. Such second messenger systems includebut are not limited to, cAMP guanylate cyclase, ion channels orphosphoinositide hydrolysis.

[0089] Examples of antagonist compounds include antibodies, or in somecases, oligonucleotides, which bind to the receptor for the polypeptideof the present invention but elicit no second messenger response or bindto the FGF-15 polypeptide itself. Alternatively, a potential antagonistmay be a mutant form of the polypeptide which binds to the receptors,however, no second messenger response is elicited and, therefore, theaction of the polypeptide is effectively blocked.

[0090] Another antagonist compound to the FGF-15 gene and gene productis an antisense construct prepared using antisense technology. Antisensetechnology can be used to control gene expression through triple-helixformation or antisense DNA or RNA, both of which methods are based onbinding of a polynucleotide to DNA or RNA. For example, the 5′ codingportion of the polynucleotide sequence, which encodes for the maturepolypeptides of the present invention, is used to design an antisenseRNA oligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription (triple helix—see Lee et al., Nucl. AcidsRes., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervanet al., Science, 251: 1360 (1991)), thereby preventing transcription andthe production of the polypeptides of the present invention. Theantisense RNA oligonucleotide hybridizes to the mRNA in vivo and blockstranslation of the mRNA molecule into the polypeptide (Antisense—Okano,J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as AntisenseInhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Theoligonucleotides described above can also be delivered to cells suchthat the antisense RNA or DNA may be expressed in vivo to inhibitproduction of the polypeptide.

[0091] Potential antagonist compounds also include small molecules whichbind to and occupy the binding site of the receptors thereby making thereceptor inaccessible to its polypeptide such that normal biologicalactivity is prevented. Examples of small molecules include, but are notlimited to, small peptides or peptide-like molecules.

[0092] Antagonist compounds may be employed to inhibit the cell growthand proliferation effects of the polypeptides of the present inventionon neoplastic cells and tissues, i.e. stimulation of angiogenesis oftumors, and, therefore, retard or prevent abnormal cellular growth andproliferation, for example, in tumor formation or growth.

[0093] The antagonists may also be employed to prevent hyper-vasculardiseases, and prevent the proliferation of epithelial lens cells afterextracapsular cataract surgery. Prevention of the mitogenic activity ofthe polypeptides of the present invention may also be desirous in casessuch as restenosis after balloon angioplasty.

[0094] The antagonists may also be employed to prevent the growth ofscar tissue during wound healing.

[0095] The antagonists may be employed in a composition with apharmaceutically acceptable carrier, e.g., as hereinafter described.

[0096] The polypeptides, agonists and antagonists of the presentinvention may be employed in combination with a suitable pharmaceuticalcarrier to comprise a pharmaceutical composition for parenteraladministration. Such compositions comprise a therapeutically effectiveamount of the polypeptide, agonist or antagonist and a pharmaceuticallyacceptable carrier or excipient. Such a carrier includes but is notlimited to saline, buffered saline, dextrose, water, glycerol, ethanol,and combinations thereof. The formulation should suit the mode ofadministration.

[0097] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. Inaddition, the polypeptides, agonists and antagonists of the presentinvention may be employed in conjunction with other therapeuticcompounds.

[0098] The pharmaceutical compositions may be administered in aconvenient manner such as by the oral, topical, intravenous,intraperitoneal, intramuscular, subcutaneous, intranasal or intradermalroutes. The pharmaceutical compositions are administered in an amountwhich is effective for treating and/or prophylaxis of the specificindication. In general, they are administered in an amount of at leastabout 10 μg/kg body weight and in-most cases they will be administeredin an amount not in excess of about 8 mg/Kg body weight per day. In mostcases, the dosage is from about 10 μg/kg to about 1 mg/kg body weightdaily, taking into account the routes of administration, symptoms, etc.In the specific case of topical administration, dosages are preferablyadministered from about 0.1 μg to 9 mg per cm².

[0099] The polypeptide of the invention and agonist and antagonistcompounds which are polypeptides, may also be employed in accordancewith the present invention by expression of such polypeptide in vivo,which is often referred to as “gene therapy.”

[0100] Thus, for example, cells may be engineered with a polynucleotide(DNA or RNA) encoding for the polypeptide ex vivo, the engineered cellsare then provided to a patient to be treated with the polypeptide. Suchmethods are well-known in the art. For example, cells may be engineeredby procedures known in the art by use of a retroviral particlecontaining RNA encoding for the polypeptide of the present invention.

[0101] Similarly, cells may be engineered in vivo for expression of thepolypeptide in vivo, for example, by procedures known in the art. Asknown in the art, a producer cell for producing a retroviral particlecontaining RNA encoding the polypeptide of the present invention may beadministered to a patient for engineering cells in vivo and expressionof the polypeptide in vivo. These and other methods for administering apolypeptide of the present invention by such methods should be apparentto those skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retroviral particle, for example, an adenovirus, which may beused to engineer cells in vivo after combination with a suitabledelivery vehicle.

[0102] Retroviruses from which the retroviral plasmid vectorshereinabove mentioned may be derived include, but are not limited to,Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses suchas Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus,gibbon ape leukemia virus, human immunodeficiency virus, adenovirus,Myeloproliferative Sarcoma Virus, and mammary tumor virus. In oneembodiment, the retroviral plasmid vector is derived from Moloney MurineLeukemia Virus.

[0103] The vector includes one or more promoters. Suitable promoterswhich may be employed include, but are not limited to, the retroviralLTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoterdescribed in Miller, et al., Biotechniques, Vol. 7, No. 9, 980-990(1989), or any other promoter (e.g., cellular promoters such aseukaryotic cellular promoters including, but not limited to, thehistone, pol III, and β-actin promoters). Other viral promoters whichmay be employed include, but are not limited to, adenovirus promoters,thymidine kinase (TK) promoters, and B19 parvovirus promoters. Theselection of a suitable promoter will be apparent to those skilled inthe art from the teachings contained herein.

[0104] The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orhetorologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the β-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

[0105] The retroviral plasmid vector is employed to transduce packagingcell lines to form producer cell lines. Examples of packaging cellswhich may be transfected include, but are not limited to, the PE501,PA317, ψ-2, ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP+E−86,GP+envAm12, and DAN cell lines as described in Miller, Human GeneTherapy, Vol. 1, pgs. 5-14 (1990), which is incorporated herein byreference in its entirety. The vector may transduce the packaging cellsthrough any means known in the art. Such means include, but are notlimited to, electroporation, the use of liposomes, and CaPO₄precipitation. In one alternative, the retroviral plasmid vector may beencapsulated into a liposome, or coupled to a lipid, and thenadministered to a host.

[0106] The producer cell line generates infectious retroviral vectorparticles which include the nucleic acid sequence(s) encoding thepolypeptides. Such retroviral vector particles then may be employed, totransduce eukaryotic cells, either in vitro or in vivo. The transducedeukaryotic cells will express the nucleic acid sequencers) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

[0107] This invention is also related to the use of the genes of thepresent invention as part of a diagnostic assay for detecting diseasesor susceptibility to diseases related to the presence of mutations inthe nucleic acid sequences encoding the polypeptide of the presentinvention.

[0108] Individuals carrying mutations in a gene of the present inventionmay be detected at the DNA level by a variety of techniques. Nucleicacids for diagnosis may be obtained from a patient's cells, such as fromblood, urine, saliva, tissue biopsy and autopsy material. The-genomicDNA may be used directly for detection or may be amplified enzymaticallyby using PCR (Saiki et al., Nature, 324:163-166 (1986)) prior toanalysis. RNA or cDNA may also be used for the same purpose. As anexample, PCR primers complementary to the nucleic acid encoding apolypeptide of the present invention can be used to identify and analyzemutations. For example, deletions and insertions can be detected by achange in size of the amplified product in comparison to the normalgenotype. Point mutations can be identified by hybridizing amplified DNAto radiolabeled RNA or alternatively, radiolabeled antisense DNAsequences. Perfectly matched sequences can be distinguished frommismatched duplexes by RNase A digestion or by differences in meltingtemperatures.

[0109] Genetic testing based on DNA sequence differences may be achievedby detection of alteration in electrophoretic mobility of DNA fragmentsin gels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g, Myerset al., Science, 230:1242 (1985)).

[0110] Sequence changes at specific locations may also be revealed bynuclease protection assays, such as RNase and S1 protection or thechemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401(1985)).

[0111] Thus the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

[0112] In addition to more conventional gel-electrophoresis and DNAsequencing, mutations can also be detected by in situ analysis.

[0113] The present invention also relates to a diagnostic assay fordetecting altered levels of FGF-15 proteins in various tissues since anover-expression of the proteins compared to normal control tissuesamples may detect the presence of abnormal cellular proliferation, forexample, a tumor. Assays used to detect levels of protein in a samplederived from a host are well-known to those of skill in the art andinclude radioimmunoassays, competitive-binding assays, Western Blotanalysis, ELISA assays and “sandwich” assay. An ELISA assay (Coligan, etal., Current Protocols in Immunology, 1(2), Chapter 6, (1991)) initiallycomprises preparing an antibody specific to an antigen to thepolypeptides of the present invention, preferably a monoclonal antibody.In addition a reporter antibody is prepared against the monoclonalantibody. To the reporter antibody is attached a detectable reagent suchas radioactivity, fluorescence or, in this example, a horseradishperoxidase enzyme. A sample is removed from a host and incubated on asolid support, e.g. a polystyrene dish, that binds the proteins in thesample. Any free protein binding sites on the dish are then covered byincubating with a non-specific protein like bovine serum albumen. Next,the monoclonal antibody is incubated in the dish during which time themonoclonal antibodies attach to any polypeptides of the presentinvention attached to the polystyrene dish. All unbound monoclonalantibody is washed out with buffer. The reporter antibody linked tohorseradish peroxidase is now placed in the dish resulting in binding ofthe reporter antibody to any monoclonal antibody bound to the protein ofinterest.

[0114] Unattached reporter antibody is then washed out. Peroxidasesubstrates are then added to the dish and the amount of color developedin a given time period is a measurement of the amount of a polypeptideof the present invention present in a given volume of patient samplewhen compared against a standard curve.

[0115] A competition assay may be employed wherein antibodies specificto a polypeptide of the present invention are attached to a solidsupport and labeled FGF-13 and a sample derived from the host are passedover the solid support and the amount of label detected, for example byliquid scintillation chromatography, can be correlated to a quantity ofa polypeptide of the present invention in the sample.

[0116] A “sandwich” assay is similar to an ELISA assay. In a “sandwich”assay a polypeptide of the present invention is passed over a solidsupport and binds to antibody attached to a solid support. A secondantibody is then bound to the polypeptide of interest. A third antibodywhich is labeled and specific to the second antibody is then passed overthe solid support and binds to the second antibody and an amount canthen be quantified.

[0117] The sequences of the present invention are also valuable forchromosome identification. The sequence is specifically targeted to andcan hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome. Few chromosome marking reagents based on actualsequence data (repeat polymorphism's) are presently available formarking chromosomal location. The mapping of DNAs to chromosomesaccording to the present invention is an important first step incorrelating those sequences with genes associated with disease.

[0118] Briefly, sequences can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3′untranslated region is used to rapidly select primers that do not spanmore than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

[0119] PCR mapping of somatic cell hybrids is a rapid procedure forassigning a particular DNA to a particular chromosome. Using the presentinvention with the same oligonucleotide primers, sublocalization can beachieved with panels of fragments from specific chromosomes or pools oflarge genomic clones in an analogous manner. Other mapping strategiesthat can similarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

[0120] Fluorescence in situ hybridization (FISH) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 50 or 60 bases. For a review of this technique, see Verma etal., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press,New York (1988).

[0121] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data (Such data are found, for example, inV. McKusick, Mendelian Inheritance in Man (available on line throughJohns Hopkins University Welch Medical Library). The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through linkage analysis (coinheritance ofphysically adjacent genes).

[0122] Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

[0123] With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

[0124] The polypeptides, their fragments or other derivatives, oranalogs thereof, or cells expressing them can be used as an immunogen toproduce antibodies thereto. These antibodies can be, for example,polyclonal or monoclonal antibodies. The present invention also includeschimeric, single chain, and humanized antibodies, as well as Fabfragments, or the product of an Fab expression library. Variousprocedures known in the art may be used for the production of suchantibodies and fragments.

[0125] Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

[0126] For preparation of monoclonal antibodies, any technique whichprovides antibodies produced by continuous cell line cultures can beused. Examples include the hybridoma technique (Kohler and Milstein,1975, Nature, 256:495-497), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), andthe EBV-hybridoma technique to produce human monoclonal antibodies(Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, Inc., pp. 77-96).

[0127] Techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produce singlechain antibodies to immunogenic polypeptide products of this invention.Also, transgenic mice may be used to express humanized antibodies toimmunogenic polypeptide products of this invention.

[0128] The present invention will be further described with reference tothe following examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

[0129] In order to facilitate understanding of the following examples,certain frequently occurring methods and/or terms will be described.

[0130] “Plasmids” are designated by a lower case p preceded and/orfollowed by capital letters and/or numbers. The starting plasmids hereinare either commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accord withpublished procedures. In addition, equivalent plasmids to thosedescribed are known in the art and will be apparent to the ordinarilyskilled artisan.

[0131] “Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

[0132] Size separation of the cleaved fragments is performed using 8percent polyacrylamide gel described by Goeddel, D. et al., NucleicAcids Res., 8:4057 (1980).

[0133] “Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

[0134] “Ligation” refers to the process of forming phosphodiester bondsbetween two double stranded nucleic acid fragments (Maniatis, T. et al.,Id., p. 146). Unless otherwise provided, ligation may be accomplishedusing known buffers and conditions with 10 units of T4 DNA ligase(“ligase”) per 0.5 μg of approximately equimolar amounts of the DNAfragments to be ligated.

[0135] Unless otherwise stated, transformation was performed asdescribed by the method of Graham, F. and Van der Eb, A., Virology,52:456-457 (1973).

EXAMPLE 1 Bacterial Expression and Purification of FGF-15 protein

[0136] The DNA sequence encoding FGF-15 ATCC # 97146, is initiallyamplified using PCR oligonucleotide primers corresponding to the 5′sequences of the processed protein (minus the signal peptide sequence)and the vector sequences 3′ to the gene. Additional nucleotidescorresponding to the gene are added to the 5′ and 3′ sequences. The 5′oligonucleotide primer has the sequence 5′ GCCAGACCATGGTAAAACCGGTGCCCCTC3′ (SEQ ID NO:3) and contains an NcoI restriction enzyme site (in bold).The 3′ sequence 5′ GGCAGGAGATCTTGTTGTCTTACTCTTGTTGAC 3′ (SEQ ID NO:4)contains complementary sequences to a Bg1II site (in bold) and isfollowed by 21 nucleotides of FGF-15 coding sequence.

[0137] The restriction enzyme sites correspond to the restriction enzymesites on the bacterial expression vector pQE60 (Qiagen, Inc. Chatsworth,Calif. 91311). pQE-60 encodes antibiotic resistance (Amp^(r)), abacterial origin of replication (ori), an IPTG-regulatable promoteroperator (P/O), a ribosome binding site (RBS), a 6-His tag andrestriction enzyme sites. pQE-60 was then digested with NcoI and Bg1II.The amplified sequences are ligated into pQE-60 and are inserted inframe with the sequence encoding for the histidine tag and the ribosomebinding site (RBS). The ligation mixture is then used to transform E.coli strain M15/rep 4 (Qiagen, Inc.) by the procedure described inSambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold SpringLaboratory Press, (1989) M15/rep4 contains multiple copies of theplasmid pREP4, which expresses the lacI repressor and also conferskanamycin resistance (Kan^(r)) Transformants are identified by theirability to grow on LB plates and ampicillin/kanamycin resistant colonieswere selected. Plasmid DNA is isolated and confirmed by restrictionanalysis. Clones containing the desired constructs are grown overnight(O/N) in liquid culturein LB media supplemented with both Amp (100ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a largeculture at a ratio of 1:100 to 1:250. The cells are grown to an opticaldensity 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG(“Isopropyl-B-D-thiogalacto pyranoside”) is then added to a finalconcentration of 1 mM. IPTG induces by inactivating the lacI repressor,clearing the P/O leading to increased gene expression. Cells are grownan extra 3 to 4 hours. Cells are then harvested by centrifugation. Thecell pellet is solubilized in the chaotropic agent 6 Molar GuanidineHCl. After clarification, solubilized FGF-15 is purified from thissolution by chromatography on a Nickel-Chelate column under conditionsthat allow for tight binding by proteins containing the 6-His tag(Hochuli, E. et al., J. Chromatography 411:177-184 (1984)). The proteinsare eluted from the column in 6 molar guanidine HCl pH 5.0 and for thepurpose of renaturation adjusted to 3 molar guanidine HCl, 100 mM sodiumphosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione(oxidized). After incubation in this solution for 12 hours the proteinsare dialyzed to 10 mmolar sodium phosphate.

EXAMPLE 2 Cloning and expression of FGF-15 using the baculovirusexpression system

[0138] The DNA sequence encoding the full length FGF-15 protein, ATCC #97146, is amplified using PCR oligonucleotide primers corresponding tothe 5′ and 3′ sequences of the gene:

[0139] The FGF-15 5′ primer has the sequence 5′ CTAGTGGATCCGCCATCATGGTAAAACCGGTGCCC 3′ (SEQ ID NO:5) and contains a BamHI restrictionenzyme site (in bold) followed by 4 nucleotides resembling an efficientsignal for the initiation of translation in eukaryotic cells (Kozak, M.,J. Mol. Biol., 196:947-950 (1987) which is just behind the first 18nucleotides of the gene (the initiation codon for translation “ATG” isunderlined).

[0140] The 3′ primer has the sequence 5′ CGACTGGTACCAGCCACGGAGCAGGAATGTCT 3′ (SEQ ID NO:6) and contains the cleavage site for therestriction endonuclease Asp718 (in bold) and 21 nucleotidescomplementary to the 3′ non-translated sequence of the gene.

[0141] The amplified sequences are isolated from a 1% agarose gel usinga commercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment is then digested with the respective endonucleasesand purified again on a 1% agarose gel. This fragment is designated F2.

[0142] The vector pA2 (modifications of pVL941 vector, discussed below)is used for the expression of the proteins using the baculovirusexpression system (for review see: Summers, M. D. and Smith, G. E. 1987,A manual of methods for baculovirus vectors and insect cell cultureprocedures, Texas Agricultural Experimental Station Bulletin No. 1555).This expression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed bythe recognition sites for the restriction endonucleases BamHI and XbaI.The polyadenylation site of the simian virus (SV)40 is used forefficient polyadenylation. For an easy selection of recombinant virusthe beta-galactosidase gene from E.coli is inserted in the sameorientation as the polyhedrin promoter followed by the polyadenylationsignal of the polyhedrin gene. The polyhedrin sequences are flanked atboth sides by viral sequences for the cell-mediated homologousrecombination of co-transfected wild-type viral DNA. Many otherbaculovirus vectors could be used in place of pA2 such as pRG1, pAc373,pVL941 and pacIM1 (Luckow, V. A. and Summers, M. D., Virology,170:31-39).

[0143] The plasmid is digested with the restriction enzymes anddephosphorylated using calf intestinal phosphatase by procedures knownin the art. The DNA is then isolated from a 1% agarose gel using thecommercially available kit (“Geneclean” BIO 101 Inc., La Jolla, Calif.).This vector DNA is designated V2.

[0144] Fragment F2 and the dephosphorylated plasmid V2 are ligated withT4 DNA ligase. E.coli DH5α cells are then transformed and bacteriaidentified that contained the plasmid (pBacFGF-15) using the respectiverestriction enzymes. The sequence of the cloned fragment are confirmedby DNA sequencing.

[0145] 5 μg of the plasmid pBacFGF-15 is co-transfected with 1.0 μg of acommercially available linearized baculovirus (“BaculoGold™ baculovirusDNA”, Pharmingen, San Diego, Calif.) using the lipofection method(Felgner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).

[0146] 1 μg of BaculoGold™ virus DNA and 5 μg of the plasmid is mixed ina sterile well of microtiter plates containing 50 μl of serum freeGrace's medium (Life Technologies Inc., Gaithersburg, Md.). Afterwards10 μl Lipofectin plus 90 μl Grace's medium are added, mixed andincubated for 15 minutes at room temperature. Then the transfectionmixture is added drop-wise to the Sf9 insect cells (ATCC CRL 1711)seeded in 35 mm tissue culture plates with 1 ml Grace's medium withoutserum. The plates are rocked back and forth to mix the newly addedsolution. The plates are then incubated for 5 hours at 27° C. After 5hours the transfection solution is removed from the plate and 1 ml ofGrace's insect medium supplemented with 10% fetal calf serum is added.The plates are put back into an incubator and cultivation continued at27° C. for four days.

[0147] After four days the supernatant is collected and plaque assaysperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with “Blue Gal” (Life Technologies Inc.,Gaithersburg) is used which allows an easy isolation of blue stainedplaques. (A detailed description of a “plaque assay” can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

[0148] Four days after the serial dilution the virus is added to thecells and blue stained plaques are picked with the tip of an Eppendorfpipette. The agar containing the recombinant viruses is then resuspendedin an Eppendorf tube containing 200 μl of Grace's medium. The agar isremoved by a brief centrifugation and the supernatant containing therecombinant baculovirus is used to infect Sf9 cells seeded in 35 mmdishes. Four days later the supernatants of these culture dishes areharvested and then stored at 4° C.

[0149] Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbaculovirus V-FGF-15 at a multiplicity of infection (MOI) of 2. Sixhours later the medium is removed and replaced with SF900 II mediumminus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42hours later 5 μCi of ³⁵S-methionine and 5 μCi ³⁵S cysteine (Amersham)are added. The cells are further incubated for 16 hours before they areharvested by centrifugation and the labelled proteins visualized bySDS-PAGE and autoradiography.

EXAMPLE 4 Expression of Recombinant FGF-15 in COS cells

[0150] The expression of plasmids, FGF-15-HA derived from a vectorpcDNA3/Amp (Ilvitrogen) containing; 1) SV40 origin of replication, 2)ampicillin resistance gene, 3) E.coli replication origin, 4) CMVpromoter followed by a polylinker region, an SV40 intron andpolyadenylation site. DNA fragments encoding the entire FGF-15 precursorand an HA tag fused in frame to the 3′ end is cloned into the polylinkerregion of the vector, therefore, the recombinant protein expression isdirected under the CMV promoter. The HA tag corresponds to an epitopederived from the influenza hemagglutinin protein as previously described(I. Wilson, H. Niman, R. Heighten, A Cherenson, M. Connolly, and R.Lerner, 1984, Cell 37:767, (1984)). The infusion of HA tag to the targetprotein allows easy detection of the recombinant protein with anantibody that recognizes the HA epitope.

[0151] The plasmid construction strategy is described as follows:

[0152] The DNA sequence encoding FGF-15, ATCC # 97146, is constructed byPCR using two primers: the 5′ primer 5′ CTAGTGGATCCGCCATCATGGTAAAACCGGTGCCC 3′ (SEQ ID NO:7) contains a BamHI sitefollowed by 18 nucleotides of coding sequence starting from theinitiation codon; the 3′ sequence 5′ GTCGACCTCGAGTGTGTGCTTACTCTTGTT 3′(SEQ ID NO:8) contains complementary sequences to an XhoI site,translation stop codon, HA tag and the last 18 nucleotides of the FGF-15coding sequence (not including the stop codon). Therefore, the PCRproduct contains a BamHI site, coding sequence followed by HA tag fusedin frame, a translation termination stop codon next to the HA tag, andan XhoI site.

[0153] The PCR amplified DNA fragments and the vector, pcDNA3/Amp, aredigested with the respective restriction enzymes and ligated. Theligation mixture is transformed into E. coli strain SURE (available fromStratagene Cloning Systems, La Jolla, Calif. 92037) the transformedculture is plated on ampicillin media plates and resistant colonies areselected. Plasmid DNA is isolated from transformants and examined byrestriction analysis for the presence of the correct fragment. Forexpression of the recombinant FGF-15 COS cells are transfected with theexpression vector by DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T.Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring LaboratoryPress, (1989)). The expression of the FGF-15-HA protein is detected byradiolabelling and immunoprecipitation method (E. Harlow, D. Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,(1988)). Cells are labelled for 8 hours with ³⁵S-cysteine two days posttransfection. Culture media is then collected and cells are lysed withdetergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5%DOC, 50 mM Tris, pH 7.5) (Wilson, I. et al., Id 37:767 (1984)). Bothcell lysate and culture media are precipitated with an HA specificmonoclonal antibody. Proteins precipitated are analyzed on 15% SDS-PAGEgels.

[0154] Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

1 32 1 759 DNA Homo sapiens CDS (1)..(759) 1 atg gta aaa ccg gtg ccc ctcttc agg aga act gat ttc aaa tta tta 48 Met Val Lys Pro Val Pro Leu PheArg Arg Thr Asp Phe Lys Leu Leu 1 5 10 15 tta tgc aac cac aag gat ctcttc ttt ctc agg gtg tct aag ctg ctg 96 Leu Cys Asn His Lys Asp Leu PhePhe Leu Arg Val Ser Lys Leu Leu 20 25 30 gat tgc ttt tcg ccc aaa tca atgtgg ttt ctt tgg aac att ttc agc 144 Asp Cys Phe Ser Pro Lys Ser Met TrpPhe Leu Trp Asn Ile Phe Ser 35 40 45 aaa gga acg cat atg ctg cag tgt ctttgt ggc aag agt ctt aag aaa 192 Lys Gly Thr His Met Leu Gln Cys Leu CysGly Lys Ser Leu Lys Lys 50 55 60 aac aag aac cca act gat ccc cag ctc aagggt ata gtg acc agg tta 240 Asn Lys Asn Pro Thr Asp Pro Gln Leu Lys GlyIle Val Thr Arg Leu 65 70 75 80 tat tgc agg caa ggc tac tac ttg caa atgcac ccc gat gga gct ctc 288 Tyr Cys Arg Gln Gly Tyr Tyr Leu Gln Met HisPro Asp Gly Ala Leu 85 90 95 gat gga acc aag ggt gac agc act aat tct acactc ttc aac ctc ata 336 Asp Gly Thr Lys Gly Asp Ser Thr Asn Ser Thr LeuPhe Asn Leu Ile 100 105 110 cca gtg gga cta cgt gtt gtt gcc atc cag ggagtg aaa aca ggg ttg 384 Pro Val Gly Leu Arg Val Val Ala Ile Gln Gly ValLys Thr Gly Leu 115 120 125 tat ata acc atg aat gga gaa ggt tac ctc taccca tca gaa ctt ttt 432 Tyr Ile Thr Met Asn Gly Glu Gly Tyr Leu Tyr ProSer Glu Leu Phe 130 135 140 acc cct gaa tgc aag ttt aaa gaa tct gtt tttgaa aat tat tat gta 480 Thr Pro Glu Cys Lys Phe Lys Glu Ser Val Phe GluAsn Tyr Tyr Val 145 150 155 160 atc tac tca tcc atg ttg tac aga caa caggaa tct ggt aga gcc tgg 528 Ile Tyr Ser Ser Met Leu Tyr Arg Gln Gln GluSer Gly Arg Ala Trp 165 170 175 ttt ttg gga tta aat aag gaa ggg caa gctatg aaa ggg aac aga gta 576 Phe Leu Gly Leu Asn Lys Glu Gly Gln Ala MetLys Gly Asn Arg Val 180 185 190 aag aaa acc aaa cca gca gct cat ttt ctaccc aag cca ttg gaa gtt 624 Lys Lys Thr Lys Pro Ala Ala His Phe Leu ProLys Pro Leu Glu Val 195 200 205 gcc atg tac cga gaa cca tct ttg cat gatgtt ggg gaa acg gtc ccg 672 Ala Met Tyr Arg Glu Pro Ser Leu His Asp ValGly Glu Thr Val Pro 210 215 220 aag cct ggg gtg acg cca agt aaa agc acaagt gcg tct gca ata atg 720 Lys Pro Gly Val Thr Pro Ser Lys Ser Thr SerAla Ser Ala Ile Met 225 230 235 240 aat gga ggc aaa cca gtc aac aag agtaag aca aca tag 759 Asn Gly Gly Lys Pro Val Asn Lys Ser Lys Thr Thr 245250 2 252 PRT Homo sapiens 2 Met Val Lys Pro Val Pro Leu Phe Arg Arg ThrAsp Phe Lys Leu Leu 1 5 10 15 Leu Cys Asn His Lys Asp Leu Phe Phe LeuArg Val Ser Lys Leu Leu 20 25 30 Asp Cys Phe Ser Pro Lys Ser Met Trp PheLeu Trp Asn Ile Phe Ser 35 40 45 Lys Gly Thr His Met Leu Gln Cys Leu CysGly Lys Ser Leu Lys Lys 50 55 60 Asn Lys Asn Pro Thr Asp Pro Gln Leu LysGly Ile Val Thr Arg Leu 65 70 75 80 Tyr Cys Arg Gln Gly Tyr Tyr Leu GlnMet His Pro Asp Gly Ala Leu 85 90 95 Asp Gly Thr Lys Gly Asp Ser Thr AsnSer Thr Leu Phe Asn Leu Ile 100 105 110 Pro Val Gly Leu Arg Val Val AlaIle Gln Gly Val Lys Thr Gly Leu 115 120 125 Tyr Ile Thr Met Asn Gly GluGly Tyr Leu Tyr Pro Ser Glu Leu Phe 130 135 140 Thr Pro Glu Cys Lys PheLys Glu Ser Val Phe Glu Asn Tyr Tyr Val 145 150 155 160 Ile Tyr Ser SerMet Leu Tyr Arg Gln Gln Glu Ser Gly Arg Ala Trp 165 170 175 Phe Leu GlyLeu Asn Lys Glu Gly Gln Ala Met Lys Gly Asn Arg Val 180 185 190 Lys LysThr Lys Pro Ala Ala His Phe Leu Pro Lys Pro Leu Glu Val 195 200 205 AlaMet Tyr Arg Glu Pro Ser Leu His Asp Val Gly Glu Thr Val Pro 210 215 220Lys Pro Gly Val Thr Pro Ser Lys Ser Thr Ser Ala Ser Ala Ile Met 225 230235 240 Asn Gly Gly Lys Pro Val Asn Lys Ser Lys Thr Thr 245 250 3 29 DNAHomo sapiens 3 gccagaccat ggtaaaaccg gtgcccctc 29 4 33 DNA Homo sapiens4 ggcaggagat cttgttgtct tactcttgtt gac 33 5 35 DNA Homo sapiens 5ctagtggatc cgccatcatg gtaaaaccgg tgccc 35 6 32 DNA Homo sapiens 6cgactggtac cagccacgga gcaggaatgt ct 32 7 35 DNA Homo sapiens 7ctagtggatc cgccatcatg gtaaaaccgg tgccc 35 8 30 DNA Homo sapiens 8gtcgacctcg agtgtgtgct tactcttgtt 30 9 155 PRT Homo sapiens 9 Met Ala GluGly Glu Ile Thr Thr Phe Thr Ala Leu Thr Glu Lys Phe 1 5 10 15 Asn LeuPro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser 20 25 30 Asn GlyGly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp Gly 35 40 45 Thr ArgAsp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala Glu 50 55 60 Ser ValGly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr Leu 65 70 75 80 AlaMet Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu 85 90 95 GluCys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr 100 105 110Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys 115 120125 Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys Ala 130135 140 Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 145 150 155 10 155PRT Homo sapiens 10 Met Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu ProGlu Asp Gly 1 5 10 15 Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys AspPro Lys Arg Leu 20 25 30 Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg Ile HisPro Asp Gly Arg 35 40 45 Val Asp Gly Val Arg Glu Lys Ser Asp Pro His IleLys Leu Gln Leu 50 55 60 Gln Ala Glu Glu Arg Gly Val Val Ser Ile Lys GlyVal Cys Ala Asn 65 70 75 80 Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg LeuLeu Ala Ser Lys Cys 85 90 95 Val Thr Asp Glu Cys Phe Phe Phe Glu Arg LeuGlu Ser Asn Asn Tyr 100 105 110 Asn Thr Tyr Arg Ser Arg Lys Tyr Thr SerTrp Tyr Val Ala Leu Lys 115 120 125 Arg Thr Gly Gln Tyr Lys Leu Gly SerLys Thr Gly Pro Gly Gln Lys 130 135 140 Ala Ile Leu Phe Leu Pro Met SerAla Lys Ser 145 150 155 11 239 PRT Homo sapiens 11 Met Gly Leu Ile TrpLeu Leu Leu Leu Ser Leu Leu Glu Pro Gly Trp 1 5 10 15 Pro Ala Ala GlyPro Gly Ala Arg Leu Arg Arg Asp Ala Gly Gly Arg 20 25 30 Gly Gly Val TyrGlu His Leu Gly Gly Ala Pro Arg Arg Arg Lys Leu 35 40 45 Tyr Cys Ala ThrLys Tyr His Leu Gln Leu His Pro Ser Gly Arg Val 50 55 60 Asn Gly Ser LeuGlu Asn Ser Ala Tyr Ser Ile Leu Glu Ile Thr Ala 65 70 75 80 Val Glu ValGly Ile Val Ala Ile Arg Gly Leu Phe Ser Gly Arg Tyr 85 90 95 Leu Ala MetAsn Lys Arg Gly Arg Leu Tyr Ala Ser Glu His Tyr Ser 100 105 110 Ala GluCys Glu Phe Val Glu Arg Ile His Glu Leu Gly Tyr Asn Thr 115 120 125 TyrAla Ser Arg Leu Tyr Arg Thr Val Ser Ser Thr Pro Gly Ala Arg 130 135 140Arg Gln Pro Ser Ala Glu Arg Leu Trp Tyr Val Ser Val Asn Gly Lys 145 150155 160 Gly Arg Pro Arg Arg Gly Phe Lys Thr Arg Arg Thr Gln Lys Ser Ser165 170 175 Leu Phe Leu Pro Arg Val Leu Asp His Arg Asp His Glu Met ValArg 180 185 190 Gln Leu Gln Ser Gly Leu Pro Arg Pro Pro Gly Lys Gly ValGln Pro 195 200 205 Arg Arg Arg Arg Gln Lys Gln Ser Pro Asp Asn Leu GluPro Ser His 210 215 220 Val Gln Ala Ser Arg Leu Gly Ser Gln Leu Glu AlaSer Ala His 225 230 235 12 206 PRT Homo sapiens 12 Met Ser Gly Pro GlyThr Ala Ala Val Ala Leu Leu Pro Ala Val Leu 1 5 10 15 Leu Ala Leu LeuAla Pro Trp Ala Gly Arg Gly Gly Ala Ala Ala Pro 20 25 30 Thr Ala Pro AsnGly Thr Leu Glu Ala Glu Leu Glu Arg Arg Trp Glu 35 40 45 Ser Leu Val AlaLeu Ser Leu Ala Arg Leu Pro Val Ala Ala Gln Pro 50 55 60 Lys Glu Ala AlaVal Gln Ser Gly Ala Gly Asp Tyr Leu Leu Gly Ile 65 70 75 80 Lys Arg LeuArg Arg Leu Tyr Cys Asn Val Gly Ile Gly Phe His Leu 85 90 95 Gln Ala LeuPro Asp Gly Arg Ile Gly Gly Ala His Ala Asp Thr Arg 100 105 110 Asp SerLeu Leu Glu Leu Ser Pro Val Glu Arg Gly Val Val Ser Ile 115 120 125 PheGly Val Ala Ser Arg Phe Phe Val Ala Met Ser Ser Lys Gly Lys 130 135 140Leu Tyr Gly Ser Pro Phe Phe Thr Asp Glu Cys Thr Phe Lys Glu Ile 145 150155 160 Leu Leu Pro Asn Asn Tyr Asn Ala Tyr Glu Ser Tyr Lys Tyr Pro Gly165 170 175 Met Phe Ile Ala Leu Ser Lys Asn Gly Lys Thr Lys Lys Gly AsnArg 180 185 190 Val Ser Pro Thr Met Lys Val Thr His Phe Leu Pro Arg Leu195 200 205 13 267 PRT Homo sapiens 13 Met Ser Leu Ser Phe Leu Leu LeuLeu Phe Phe Ser His Leu Ile Leu 1 5 10 15 Ser Ala Trp Ala His Gly GluLys Arg Leu Ala Pro Lys Gly Gln Pro 20 25 30 Gly Pro Ala Ala Thr Asp ArgAsn Pro Arg Gly Ser Ser Ser Arg Gln 35 40 45 Ser Ser Ser Ser Ala Met SerSer Ser Ser Ala Ser Ser Ser Pro Ala 50 55 60 Ala Ser Leu Gly Ser Gln GlySer Gly Leu Glu Gln Ser Ser Phe Gln 65 70 75 80 Trp Ser Leu Gly Ala ArgThr Gly Ser Leu Tyr Cys Arg Val Gly Ile 85 90 95 Gly Phe His Leu Gln IleTyr Pro Asp Gly Lys Val Asn Gly Ser His 100 105 110 Glu Ala Asn Met LeuSer Val Leu Glu Ile Phe Ala Val Ser Gln Gly 115 120 125 Ile Val Gly IleArg Gly Val Phe Ser Asn Lys Phe Leu Ala Met Ser 130 135 140 Lys Lys GlyLys Leu His Ala Ser Ala Lys Phe Thr Asp Asp Cys Lys 145 150 155 160 PheArg Glu Arg Phe Gln Glu Asn Ser Tyr Asn Thr Tyr Ala Ser Ala 165 170 175Ile His Arg Thr Glu Lys Thr Gly Arg Glu Trp Tyr Val Ala Leu Asn 180 185190 Lys Arg Gly Lys Ala Lys Arg Gly Cys Ser Pro Arg Val Lys Pro Gln 195200 205 His Ile Ser Thr His Phe Leu Pro Arg Phe Lys Gln Ser Glu Gln Pro210 215 220 Glu Leu Ser Phe Thr Val Thr Val Pro Glu Lys Lys Asn Pro ProSer 225 230 235 240 Pro Ile Lys Ser Lys Ile Pro Leu Ser Ala Pro Arg LysAsn Thr Asn 245 250 255 Ser Val Lys Tyr Arg Leu Lys Phe Arg Phe Gly 260265 14 208 PRT Homo sapiens 14 Met Ala Leu Gly Gln Lys Leu Phe Ile ThrMet Ser Arg Gly Ala Gly 1 5 10 15 Arg Leu Gln Gly Thr Leu Trp Ala LeuVal Phe Leu Gly Ile Leu Val 20 25 30 Gly Met Val Val Pro Ser Pro Ala GlyThr Arg Ala Asn Asn Thr Leu 35 40 45 Leu Asp Ser Arg Gly Trp Gly Thr LeuLeu Ser Arg Ser Arg Ala Gly 50 55 60 Leu Ala Gly Glu Ile Ala Gly Val AsnTrp Glu Ser Gly Tyr Leu Val 65 70 75 80 Gly Ile Lys Arg Gln Arg Arg LeuTyr Cys Asn Val Gly Ile Gly Phe 85 90 95 His Leu Gln Val Leu Pro Asp GlyArg Ile Ser Gly Thr His Glu Glu 100 105 110 Asn Pro Tyr Ser Leu Leu GluIle Ser Thr Val Glu Arg Gly Val Val 115 120 125 Ser Leu Phe Gly Val ArgSer Ala Leu Phe Val Ala Met Asn Ser Lys 130 135 140 Gly Arg Leu Tyr AlaThr Pro Ser Phe Gln Glu Glu Cys Lys Phe Arg 145 150 155 160 Glu Thr LeuLeu Pro Asn Asn Tyr Asn Ala Tyr Glu Ser Asp Leu Tyr 165 170 175 Gln GlyThr Tyr Ile Ala Leu Ser Lys Tyr Gly Arg Val Lys Arg Gly 180 185 190 SerLys Val Ser Pro Ile Met Thr Val Thr His Phe Leu Pro Arg Ile 195 200 20515 194 PRT Homo sapiens 15 Met His Lys Trp Ile Leu Thr Trp Ile Leu ProThr Leu Leu Tyr Arg 1 5 10 15 Ser Cys Phe His Ile Ile Cys Leu Val GlyThr Ile Ser Leu Ala Cys 20 25 30 Asn Asp Met Thr Pro Glu Gln Met Ala ThrAsn Val Asn Cys Ser Ser 35 40 45 Pro Glu Arg His Thr Arg Ser Tyr Asp TyrMet Glu Gly Gly Asp Ile 50 55 60 Arg Val Arg Arg Leu Phe Cys Arg Thr GlnTrp Tyr Leu Arg Ile Asp 65 70 75 80 Lys Arg Gly Lys Val Lys Gly Thr GlnGlu Met Lys Asn Asn Tyr Asn 85 90 95 Ile Met Glu Ile Arg Thr Val Ala ValGly Ile Val Ala Ile Lys Gly 100 105 110 Val Glu Ser Glu Phe Tyr Leu AlaMet Asn Lys Glu Gly Lys Leu Tyr 115 120 125 Ala Lys Lys Glu Cys Asn GluAsp Cys Asn Phe Lys Glu Leu Ile Leu 130 135 140 Glu Asn His Tyr Asn ThrTyr Ala Ser Ala Lys Trp Thr His Asn Gly 145 150 155 160 Gly Glu Met PheVal Ala Leu Asn Gln Lys Gly Ile Pro Val Arg Gly 165 170 175 Lys Lys ThrLys Lys Glu Gln Lys Thr Ala His Phe Leu Pro Met Ala 180 185 190 Ile Thr16 215 PRT Homo sapiens 16 Met Gly Ser Pro Arg Ser Ala Leu Ser Cys LeuLeu Leu His Leu Leu 1 5 10 15 Val Leu Cys Leu Gln Ala Gln Val Thr ValGln Ser Ser Pro Asn Phe 20 25 30 Thr Gln His Val Arg Glu Gln Ser Leu ValThr Asp Gln Leu Ser Arg 35 40 45 Arg Leu Ile Arg Thr Tyr Gln Leu Tyr SerArg Thr Ser Gly Lys His 50 55 60 Val Gln Val Leu Ala Asn Lys Arg Ile AsnAla Met Ala Glu Asp Gly 65 70 75 80 Asp Pro Phe Ala Lys Leu Ile Val GluThr Asp Thr Phe Gly Ser Arg 85 90 95 Val Arg Val Arg Gly Ala Glu Thr GlyLeu Tyr Ile Cys Met Asn Lys 100 105 110 Lys Gly Lys Leu Ile Ala Lys SerAsn Gly Lys Gly Lys Asp Cys Val 115 120 125 Phe Thr Glu Ile Val Leu GluAsn Asn Tyr Thr Ala Leu Gln Asn Ala 130 135 140 Lys Tyr Glu Gly Trp TyrMet Ala Phe Thr Arg Lys Gly Arg Pro Arg 145 150 155 160 Lys Gly Ser LysThr Arg Gln His Gln Arg Glu Val His Phe Met Lys 165 170 175 Arg Leu ProArg Gly His His Thr Thr Glu Gln Ser Leu Arg Phe Glu 180 185 190 Phe LeuAsn Tyr Pro Pro Phe Thr Arg Ser Leu Arg Gly Ser Gln Arg 195 200 205 ThrTrp Ala Pro Glu Pro Arg 210 215 17 208 PRT Homo sapiens 17 Met Ala ProLeu Gly Glu Val Gly Asn Tyr Phe Gly Val Gln Asp Ala 1 5 10 15 Val ProPhe Gly Asn Val Pro Val Leu Pro Val Asp Ser Pro Val Leu 20 25 30 Leu SerAsp His Leu Gly Gln Ser Glu Ala Gly Gly Leu Pro Arg Gly 35 40 45 Pro AlaVal Thr Asp Leu Asp His Leu Lys Gly Ile Leu Arg Arg Arg 50 55 60 Gln LeuTyr Cys Arg Thr Gly Phe His Leu Glu Ile Phe Pro Asn Gly 65 70 75 80 ThrIle Gln Gly Thr Arg Lys Asp His Ser Arg Phe Gly Ile Leu Glu 85 90 95 PheIle Ser Ile Ala Val Gly Leu Val Ser Ile Arg Gly Val Asp Ser 100 105 110Gly Leu Tyr Leu Gly Met Asn Glu Lys Gly Glu Leu Tyr Gly Ser Glu 115 120125 Lys Leu Thr Gln Glu Cys Val Phe Arg Glu Gln Phe Glu Glu Asn Trp 130135 140 Tyr Asn Thr Tyr Ser Ser Asn Leu Tyr Lys His Val Asp Thr Gly Arg145 150 155 160 Arg Tyr Tyr Val Ala Leu Asn Lys Asp Gly Thr Pro Arg GluGly Thr 165 170 175 Arg Thr Lys Arg His Gln Lys Phe Thr His Phe Leu ProArg Pro Val 180 185 190 Asp Pro Asp Lys Val Pro Glu Leu Tyr Lys Asp IleLeu Ser Gln Ser 195 200 205 18 181 PRT Homo sapiens 18 Met Glu Ser LysGlu Pro Gln Leu Lys Gly Ile Val Thr Arg Leu Phe 1 5 10 15 Ser Gln GlnGly Tyr Phe Leu Gln Met His Pro Asp Gly Thr Ile Asp 20 25 30 Gly Thr LysAsp Glu Asn Ser Asp Tyr Thr Leu Phe Asn Leu Ile Pro 35 40 45 Val Gly LeuArg Val Val Ala Ile Gln Gly Val Lys Ala Ser Leu Tyr 50 55 60 Val Ala MetAsn Gly Glu Gly Tyr Leu Tyr Ser Ser Asp Val Phe Thr 65 70 75 80 Pro GluCys Lys Phe Lys Glu Ser Val Phe Glu Asn Tyr Tyr Val Ile 85 90 95 Tyr SerSer Thr Leu Tyr Arg Gln Gln Glu Ser Gly Arg Ala Trp Phe 100 105 110 LeuGly Leu Asn Lys Glu Gly Gln Ile Met Lys Gly Asn Arg Val Lys 115 120 125Lys Thr Lys Pro Ser Ser His Phe Val Pro Lys Pro Ile Glu Val Cys 130 135140 Met Tyr Arg Glu Pro Ser Leu His Glu Ile Gly Glu Lys Gln Gly Arg 145150 155 160 Ser Arg Lys Ser Ser Gly Thr Pro Thr Met Asn Gly Gly Lys ValVal 165 170 175 Asn Gln Asp Ser Thr 180 19 255 PRT Homo sapiens 19 MetSer Gly Lys Val Thr Lys Pro Lys Glu Glu Lys Asp Ala Ser Lys 1 5 10 15Val Leu Asp Asp Ala Pro Pro Gly Thr Gln Glu Tyr Ile Met Leu Arg 20 25 30Gln Asp Ser Ile Gln Ser Ala Glu Leu Lys Lys Lys Glu Ser Pro Phe 35 40 45Arg Ala Lys Cys His Glu Ile Phe Cys Cys Pro Leu Lys Gln Val His 50 55 60His Lys Glu Asn Thr Glu Pro Glu Glu Pro Gln Leu Lys Gly Ile Val 65 70 7580 Thr Lys Leu Tyr Ser Arg Gln Gly Tyr His Leu Gln Leu Gln Ala Asp 85 9095 Gly Thr Ile Asp Gly Thr Lys Asp Glu Asp Ser Thr Tyr Thr Leu Phe 100105 110 Asn Leu Ile Pro Val Gly Leu Arg Val Val Ala Ile Gln Gly Val Gln115 120 125 Thr Lys Leu Tyr Leu Ala Met Asn Ser Glu Gly Tyr Leu Tyr ThrSer 130 135 140 Glu Leu Phe Thr Pro Glu Cys Lys Phe Lys Glu Ser Val PheGlu Asn 145 150 155 160 Tyr Tyr Val Thr Tyr Ser Ser Met Ile Tyr Arg GlnGln Gln Ser Gly 165 170 175 Arg Gly Trp Tyr Leu Gly Leu Asn Lys Glu GlyGlu Ile Met Lys Gly 180 185 190 Asn His Val Lys Lys Asn Lys Pro Ala AlaHis Phe Leu Pro Lys Pro 195 200 205 Leu Lys Val Ala Met Tyr Lys Glu ProSer Leu His Asp Leu Thr Glu 210 215 220 Phe Ser Arg Ser Gly Ser Gly ThrPro Thr Lys Ser Arg Ser Val Ser 225 230 235 240 Gly Val Leu Asn Gly GlyLys Ser Met Ser His Asn Glu Ser Thr 245 250 255 20 208 PRT Homo sapiens20 Met Trp Lys Trp Ile Leu Thr His Cys Ala Ser Ala Phe Pro His Leu 1 510 15 Pro Gly Cys Cys Cys Cys Cys Phe Leu Leu Leu Phe Leu Val Ser Ser 2025 30 Val Pro Val Thr Cys Gln Ala Leu Gly Gln Asp Met Val Ser Pro Glu 3540 45 Ala Thr Asn Ser Ser Ser Ser Ser Phe Ser Ser Pro Ser Ser Ala Gly 5055 60 Arg His Val Arg Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg 6570 75 80 Lys Leu Phe Ser Phe Thr Lys Tyr Phe Leu Lys Ile Glu Lys Asn Gly85 90 95 Lys Val Ser Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser Ile Leu Glu100 105 110 Ile Thr Ser Val Glu Ile Gly Val Val Ala Val Lys Ala Ile AsnSer 115 120 125 Asn Tyr Tyr Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr GlySer Lys 130 135 140 Glu Phe Asn Asn Asp Cys Lys Leu Lys Glu Arg Ile GluGlu Asn Gly 145 150 155 160 Tyr Asn Thr Tyr Ala Ser Phe Asn Trp Gln HisAsn Gly Arg Gln Met 165 170 175 Tyr Val Ala Leu Asn Gly Lys Gly Ala ProArg Arg Gly Gln Lys Thr 180 185 190 Arg Arg Lys Asn Thr Ser Ala His PheLeu Pro Met Val Val His Ser 195 200 205 21 212 PRT Homo sapiens 21 ArgLeu Leu Pro Asn Leu Thr Leu Cys Leu Gln Leu Leu Ile Leu Cys 1 5 10 15Cys Gln Thr Gln Gly Glu Asn His Pro Ser Pro Asn Phe Asn Gln Tyr 20 25 30Val Arg Asp Gln Gly Ala Met Thr Asp Gln Leu Ser Arg Arg Gln Ile 35 40 45Arg Glu Tyr Gln Leu Tyr Ser Arg Thr Ser Gly Lys His Val Gln Val 50 55 60Pro Gly Arg Arg Ile Ser Ala Thr Ala Glu Asp Gly Asn Lys Phe Ala 65 70 7580 Lys Leu Ile Val Glu Thr Asp Thr Phe Gly Ser Arg Val Arg Ile Lys 85 9095 Gly Ala Glu Ser Glu Lys Tyr Ile Cys Met Asn Lys Arg Gly Lys Leu 100105 110 Ile Gly Lys Pro Ser Gly Lys Ser Lys Asp Cys Val Phe Thr Glu Ile115 120 125 Val Leu Glu Asn Asn Tyr Thr Ala Phe Gln Asn Ala Arg His GluGly 130 135 140 Trp Phe Met Val Phe Thr Arg Gln Gly Arg Pro Arg Gln AlaSer Arg 145 150 155 160 Ser Arg Gln Asn Gln Arg Glu Ala His Phe Ile LysArg Leu Tyr Gln 165 170 175 Gly Gln Leu Pro Phe Pro Asn His Ala Glu LysGln Lys Gln Phe Glu 180 185 190 Phe Val Gly Ser Ala Pro Thr Arg Arg ThrLys Arg Thr Arg Arg Pro 195 200 205 Gln Pro Leu Thr 210 22 225 PRT Homosapiens 22 Met Ala Ala Leu Ala Ser Ser Leu Ile Arg Gln Lys Arg Glu ValArg 1 5 10 15 Glu Pro Gly Gly Ser Arg Pro Val Ser Ala Gln Arg Arg ValCys Pro 20 25 30 Arg Gly Thr Lys Ser Leu Cys Gln Lys Gln Leu Leu Ile LeuLeu Ser 35 40 45 Lys Val Arg Leu Cys Gly Gly Arg Pro Ala Arg Pro Asp ArgGly Pro 50 55 60 Glu Pro Gln Leu Lys Gly Ile Val Thr Lys Leu Phe Cys ArgGln Gly 65 70 75 80 Phe Tyr Leu Gln Ala Asn Pro Asp Gly Ser Ile Gln GlyThr Pro Glu 85 90 95 Asp Thr Ser Ser Phe Thr His Phe Asn Leu Ile Pro ValGly Leu Arg 100 105 110 Val Val Thr Ile Gln Ser Ala Lys Leu Gly His TyrMet Ala Met Asn 115 120 125 Ala Glu Gly Leu Leu Tyr Ser Ser Pro His PheThr Ala Glu Cys Arg 130 135 140 Phe Lys Glu Cys Val Phe Glu Asn Tyr TyrVal Leu Tyr Ala Ser Ala 145 150 155 160 Leu Tyr Arg Gln Arg Arg Ser GlyArg Ala Trp Tyr Leu Gly Leu Asp 165 170 175 Lys Glu Gly Gln Val Met LysGly Asn Arg Val Lys Lys Thr Lys Ala 180 185 190 Ala Ala His Phe Leu ProLys Leu Leu Glu Val Ala Met Tyr Gln Glu 195 200 205 Pro Ser Leu His SerVal Pro Glu Ala Ser Pro Ser Ser Pro Pro Ala 210 215 220 Pro 225 23 252PRT Homo sapiens 23 Met Val Lys Pro Val Pro Leu Phe Arg Arg Thr Asp PheLys Leu Leu 1 5 10 15 Leu Cys Asn His Lys Asp Leu Phe Phe Leu Arg ValSer Lys Leu Leu 20 25 30 Asp Cys Phe Ser Pro Lys Ser Met Trp Phe Leu TrpAsn Ile Phe Ser 35 40 45 Lys Gly Thr His Met Leu Gln Cys Leu Cys Gly LysSer Leu Lys Lys 50 55 60 Asn Lys Asn Pro Thr Asp Pro Gln Leu Lys Gly IleVal Thr Arg Leu 65 70 75 80 Tyr Cys Arg Gln Gly Tyr Tyr Leu Gln Met HisPro Asp Gly Ala Leu 85 90 95 Asp Gly Thr Lys Gly Asp Ser Thr Asn Ser ThrLeu Phe Asn Leu Ile 100 105 110 Pro Val Gly Leu Arg Val Val Ala Ile GlnGly Val Lys Thr Gly Leu 115 120 125 Tyr Ile Thr Met Asn Gly Glu Gly TyrLeu Tyr Pro Ser Glu Leu Phe 130 135 140 Thr Pro Glu Cys Lys Phe Lys GluSer Val Phe Glu Asn Tyr Tyr Val 145 150 155 160 Ile Tyr Ser Ser Met LeuTyr Arg Gln Gln Glu Ser Gly Arg Ala Trp 165 170 175 Phe Leu Gly Leu AsnLys Glu Gly Gln Ala Met Lys Gly Asn Arg Val 180 185 190 Lys Lys Thr LysPro Ala Ala His Phe Leu Pro Lys Pro Leu Glu Val 195 200 205 Ala Met TyrArg Glu Pro Ser Leu His Asp Val Gly Glu Thr Val Pro 210 215 220 Lys ProGly Val Thr Pro Ser Lys Ser Thr Ser Ala Ser Ala Ile Met 225 230 235 240Asn Gly Gly Lys Pro Val Asn Lys Ser Lys Thr Thr 245 250 24 155 PRT Homosapiens 24 Met Ala Leu Gly Leu Leu Thr Thr Pro Leu Ala Gly His Val ArgTyr 1 5 10 15 Asp His Leu Lys Gly Leu Gly Ile Val Arg Arg Arg Leu TyrCys Arg 20 25 30 Thr Gly Gly Phe His Leu Gln Ile Leu Pro Asp Gly Arg IleAsp Gly 35 40 45 Thr Lys Glu Asp Asn Ser Pro Tyr Ser Leu Leu Glu Leu IlePro Val 50 55 60 Glu Val Gly Val Val Ala Ile Lys Gly Val Glu Ser Gly LeuTyr Leu 65 70 75 80 Ala Met Asn Lys Lys Gly Lys Leu Tyr Ala Ser Glu LeuPhe Thr Asp 85 90 95 Glu Cys Lys Phe Lys Glu Arg Val Leu Glu Asn Asn TyrAsn Thr Tyr 100 105 110 Ala Ser Ala Leu Tyr Arg Ser Gly Arg Gly Trp TyrVal Ala Leu Asn 115 120 125 Lys Glu Gly Gln Pro Lys Lys Gly Asn Arg ValLys Lys Thr Gln Lys 130 135 140 Ala Ala His Phe Leu Pro Arg Pro Leu GluVal 145 150 155 25 17 PRT Homo sapiens SITE (2) Xaa equals any aminoacid 25 Gly Xaa Leu Xaa Ser Xaa Xaa Xaa Xaa Xaa Xaa Asp Cys Xaa Phe Xaa1 5 10 15 Glu 26 17 PRT Homo sapiens SITE (2) Xaa equals any amino acid26 Gly Xaa Leu Xaa Ser Xaa Xaa Xaa Xaa Xaa Xaa Glu Cys Xaa Phe Xaa 1 510 15 Glu 27 17 PRT Homo sapiens SITE (2) Xaa equals any amino acid 27Gly Xaa Leu Xaa Thr Xaa Xaa Xaa Xaa Xaa Xaa Asp Cys Xaa Phe Xaa 1 5 1015 Glu 28 17 PRT Homo sapiens SITE (2) Xaa equals any amino acid 28 GlyXaa Leu Xaa Thr Xaa Xaa Xaa Xaa Xaa Xaa Glu Cys Xaa Phe Xaa 1 5 10 15Glu 29 17 PRT Homo sapiens SITE (2) Xaa equals any amino acid 29 Gly XaaLeu Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Asp Cys Xaa Phe Xaa 1 5 10 15 Glu 3017 PRT Homo sapiens SITE (2) Xaa equals any amino acid 30 Gly Xaa LeuXaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Glu Cys Xaa Phe Xaa 1 5 10 15 Glu 31 17PRT Homo sapiens SITE (2) Xaa equals any amino acid 31 Gly Xaa Leu XaaGly Xaa Xaa Xaa Xaa Xaa Xaa Asp Cys Xaa Phe Xaa 1 5 10 15 Glu 32 17 PRTHomo sapiens SITE (2) Xaa equals any amino acid 32 Gly Xaa Leu Xaa GlyXaa Xaa Xaa Xaa Xaa Xaa Glu Cys Xaa Phe Xaa 1 5 10 15 Glu

What is claimed is:
 1. An isolated polynucleotide comprising a memberselected from the group consisting of: (a) a polynucleotide encoding thepolypeptide as set forth in FIG. 1; (b) a polynucleotide capable ofhybridizing to and which is at least 70% identical to the polynucleotideof (a); and (c) a polynucleotide fragment of the polynucleotide of (a)or (b).
 2. The polynucleotide of claim 1 encoding the polypeptidecomprising amino acid 1 to amino acid 252 as set forth in SEQ ID NO:2.3. The polynucleotide of claim 1 wherein the polynucleotide is DNA. 4.The polynucleotide of claim 1 wherein said polynucleotide is RNA.
 5. Thepolynucleotide of claim 1 wherein the polynucleotide is genomic DNA. 6.The polynucleotide of claim 1 comprising from nucleotide 1 to nucleotide759 as set forth in SEQ ID NO:1.
 7. An isolated polynucleotidecomprising a member selected from the group consisting of: (a) apolynucleotide encoding a mature polypeptide encoded by the DNAcontained in ATCC Deposit No. 97146; (b) a polynucleotide encoding thepolypeptide expressed by the DNA contained in ATCC Deposit No. 97146;(c) a polynucleotide capable of hybridizing to and which is at least 70%identical to the polynucleotide of (a) or (b); and (d) a polynucleotidefragment of the polynucleotide of (a), (b) or (c).
 8. The polynucleotideof claim 7 wherein said polynucleotide encodes a polypeptide expressedby the DNA contained in ATCC Deposit No.
 97146. 9. The polynucleotide ofclaim 7 wherein said polynucleotide encodes a polypeptide expressed bythe DNA contained in ATCC Deposit No.
 97146. 10. The polynucleotide ofclaim 7 wherein said polynucleotide encodes a polypeptide expressed bythe DNA contained in ATCC Deposit No.
 97146. 11. A vector containing theDNA of claim
 2. 12. A host cell genetically engineered with the vectorof claim
 11. 13. A process for producing a polypeptide comprising:expressing from the host cell of claim 12 the polypeptide encoded bysaid DNA.
 14. A process for producing cells capable of expressing apolypeptide comprising genetically engineering cells with the vector ofclaim
 11. 15. A polypeptide selected from the group consisting of (i) apolypeptide having the deduced amino acid sequence of FIG. 1 andfragments, analogs and derivatives thereof; and (ii) a polypeptideencoded by the cDNA of ATCC Deposit No. 97146 and fragments, analogs andderivatives of said polypeptide.
 16. The polypeptide of claim 15 whereinthe polypeptide has the deduced amino acid sequence of FIG.
 1. 17. Anantibody against the polypeptide of claim
 15. 18. A compound whichinhibits the polypeptide of claim
 15. 19. A compound which activates areceptor to the polypeptide of claim
 15. 20. A method for the treatmentof a patient having need of an FGF-15 polypeptide comprising:administering to the patient a therapeutically effective amount of thepolypeptide of claim
 15. 21. A method for the treatment of a patienthaving need to inhibit an FGF-15 polypeptide comprising: administeringto the patient a therapeutically effective amount of the compound ofclaim
 18. 22. The method of claim 20 wherein said therapeuticallyeffective amount of said polypeptide is administered by providing to thepatient DNA encoding said polypeptide and expressing said polypeptide invivo.
 23. The method of claim 21 wherein said compound is a polypeptideand a therapeutically effective amount of the compound is administeredby providing to the patient DNA encoding said antagonist and expressingsaid antagonist in vivo.
 24. A process for identifying compounds activeas agonists to the polypeptide of claim 15 comprising: (a) combining acompound to be screened and a reaction mixture containing cells underconditions where the cells are normally stimulated by said polypeptide,said reaction mixture containing a label incorporated into the cells asthey proliferate; and (b) determining the extent of proliferation of thecells to identify if the compound is an effective agonist.
 25. A processfor identifying compounds active as antagonists to the polypeptide ofclaim 15 comprising: (a) combining a compound to be screened, thepolypeptide and a reaction mixture containing cells under conditionswhere the cells are normally stimulated by said polypeptide, saidreaction mixture containing a label incorporated into the cells as theyproliferate; and (b) determining the extent of proliferation of thecells to identify if the compound is an effective antagonist.
 26. Aprocess for diagnosing a disease or a susceptibility to a diseaserelated to an under-expression of the polypeptide of claim 15comprising: determining a mutation in the nucleic acid sequence encodingsaid polypeptide.
 27. A diagnostic process comprising: analyzing for thepresence of the polypeptide of claim 15 in a sample derived from a host.